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United States Patent |
6,066,331
|
Barenholz
,   et al.
|
May 23, 2000
|
Method for preparation of vesicles loaded with biological structures,
biopolymers and/or oligomers
Abstract
A composition useful for preparing vesicles loaded with biological
cell-structures, biopolymers and/or -oligomers is prepared by solubilizing
amphiphatic material such as a phospholipid in a polar-protic solvent
miscible with water, solubilizing biological cell-structures, biopolymers
and/or -oligomers in an aqueous medium, mixing the polar-protic solvent
containing the amphiphatic material with the aqueous medium containing the
biological cell-structures, biopolymers and/or -oligomers, and
lyophilizing the resultant mixture to form a dry product. The dry product
is hydrated in an aqueous medium to form the loaded vesicles. The
polar-protic solvent may be tert-butanol, and the aqueous medium may
contain a salt such as sodium chloride, an isoosmotic cryoprotectant such
as lactose, sucrose or trehalose, or a mixture of the salt and the
cryoprotectant. A medicament for disease treatment is formed by mixing the
loaded vesicles with a pharmaceutically acceptable vehicle.
Inventors:
|
Barenholz; Yechezkel (18 Nevenh Shaannan St., Jerusalem, IL);
Bar; Lilianne K. (Menuha Venahala 37/7, Rehovot, IL);
Diminsky; Dvorah (Harazim 21, Jerusalem, IL);
Baru; Moshe (Hadarim Street, Pardes-Hanna, IL)
|
Appl. No.:
|
710576 |
Filed:
|
August 13, 1996 |
Current U.S. Class: |
424/450; 424/93.7; 424/94.1; 424/520; 435/177; 435/182; 436/528; 436/535; 514/2; 514/44; 530/812; 530/817 |
Intern'l Class: |
A61K 009/127; C12N 011/02; G01N 033/544; C07K 017/02 |
Field of Search: |
435/174,177,180
424/450,93.7,94.1,520
436/528,535
514/2,44
530/812,817
|
References Cited
U.S. Patent Documents
4229360 | Oct., 1980 | Schneider et al. | 260/403.
|
4235871 | Nov., 1980 | Papahadjopoulos et al. | 424/19.
|
Primary Examiner: Naff; David M.
Attorney, Agent or Firm: Jacobson, Price, Holman & Stern, PLLC
Parent Case Text
This is a continuation of application Ser. No. 08/592,437, filed Feb. 6,
1996, now abandoned, which is a national stage of PCT/EP94/02242 filed
Jul. 8, 1994.
Claims
We claim:
1. A method for preparation of a composition useful in preparing vesicles
loaded with biological cell-structures, biopolymers or -oligomers by
co-drying a fraction of amphiphatic material and a fraction of biological
cell-structures, biopolymers or -oligomers wherein said fraction of
amphiphatic material is present in an organic solvent which is miscible
with water and said fraction of biological cell-structures, biopolymers or
-oligomers is present in an aqueous medium, comprising the steps of
a) solubilizing amphiphatic material in a polar-protic solvent miscible
with water to effect fraction A,
b) solubilizing biological cell-structures, biopolymers or -oligomers or
mixture of biopolymers and oligomers in a physiologically compatible
aqueous medium, optionally having a salt content equivalent to up to a 5%
by weight sodium chloride solution, to effect fraction B,
c) mixing together the fractions A and B, and
d) lyophilizing the fraction obtained in step c).
2. A method for preparing a composition useful in preparing loaded
vesicles, from an organic-solvent fraction and an aqueous fraction,
comprising the steps of:
a) solubilizing an amphiphatic material in a polar-protic solvent miscible
with water to effect fraction A,
b) solubilizing biological cell-structures, biopolymers or -oligomers, or
combination of biopolymers and -oligomers in a physiologically compatible
aqueous medium, to effect fraction B,
c) mixing together the fractions A and B to obtain a mixed fraction, which
contains the polar-protic solvent and the aqueous medium, and
d) lyophilizing the mixed fraction to give a dry product.
3. The method of claim 2 wherein the amphiphatic substance is selected from
the group consisting of saturated and unsaturated phospholipids, and
mixtures thereof, and mixtures with cholesterol of saturated and
unsaturated phospholipids, and mixtures thereof.
4. The method of claim 3, wherein the phospholipids are hydrogenated or
non-hydrogenated soybean derived phospholipids, egg yolk phospholipids,
dimyristoyl phosphatidyl choline, dimyristoyl phosphatidyl glycerol, or
mixture thereof.
5. The method of claim 4, wherein the phospholipids comprise a mixture of
dimyristoyl phosphatidyl choline and dimyristoyl phosphatidyl glycerol at
a molar ratio of dimyristoyl phosphatidyl choline:dimyristoyl phosphatidyl
glycerol between 1:20 and 20:1.
6. The method of claim 2, wherein said polar-protic solvent is
tert-butanol.
7. The method of claim 2, wherein the biological cell-structures are
natural or transformed B-cells, ribosomes, or mitochondriae; the
biopolymers or -oligomers are enzymes, proenzymes, cofactors, virions, or
virion surface antigens, antigens, antibodies, complement factors,
hormones, nucleotides, DNA, mRNA, rRNA, tRNA, or antisense RNA.
8. The method of claim 2, wherein the physiologically compatible aqueous
medium is a solution of about 0.9% by weight sodium chloride, isoosmotic
cryoprotectant, or mixture thereof.
9. The method of claim 8, wherein the cryoprotectant is lactose, sucrose,
or trehalose.
10. The method of claim 2 further comprising the step of hydrating the dry
product in an aqueous medium to form liposomes.
11. The method of claim 2, wherein the physiologically compatible aqueous
medium has a salt content equivalent to up to a 5% by weight sodium
chloride solution.
12. The dry product made by the method of claim 2.
13. The dry product made by the method of claim 3.
14. The dry product made by the method of claim 4.
15. The dry product made by the method of claim 5.
16. The dry product made by the method of claim 6.
17. The dry product made by the method of claim 7.
18. The dry product made by the method of claim 8.
19. The dry product made by the method of claim 9.
20. The liposomes made by the method of claim 10.
21. A medicament comprising the dry product of claim 12 reconstituted with
water in combination with a pharmaceutically acceptable vehicle.
22. A medicament comprising the dry product of claim 13 reconstituted with
water in combination with a pharmaceutically acceptable vehicle.
23. A medicament comprising the liposomes of claim 20 in combination with a
pharmaceutically acceptable vehicle.
24. A method of treating disease by administering to a patient an effective
amount of the medicament according to claim 21.
25. A method of treating disease by administering to a patient an effective
amount of the medicament according to claim 22.
26. A method of treating disease by administering to a patient an effective
amount of the medicament according to claims 23.
27. Method of claim 25 wherein the effective amount is a dosage of up to
2,000 mg, measured by phospholipid per kg body wt.
Description
The invention is related to a method for preparation of vesicles loaded
with biological structures, biopolymers and/or - oligomers, a formulation
comprising vesicles loaded with biological structures, biopolymers and/or
-oligomers obtainable according to the method of the invention, a
medicament comprising the formulation of the invention as well as a method
of treating diseases administering the medicament of the invention.
Several attempts have been tried to use lipid vesicles formed by natural or
synthetic phospholipids as vehicles for the administration of effective
substances.
Grey, A. and Morgan, J. report that liposomes were first described nearly a
quarter of a century ago and have been useful models for studying the
physical chemistry of lipid bilayers and the biology of the cell membrane.
It was also realised that they might be used as vehicles for the delivery
of drugs but clinical application have been slow to emerge. Proposed
clinical uses have included vaccine adjuvancy, gene Transfer and
diagnostic imaging but the major effort has been in the development of
liposomes as targetable drug carriers in the treatment of malignancy.
Although based on good in vitro data and animal studies, the strategies
have been mostly impractical due to the predominant but unwanted uptake by
the reticuloendothelial system and the limited extent of extravasation.
The same features have nonetheless been turned to advantage in the case of
amphotericin B which has recently become the first liposomally formulated
agent to be licensed for parenteral use. Liposomal doxorubicin is
currently also being evaluated in clinical trials. The early evidence
suggests that while liposomal encapsulation may not greatly enhance their
efficacy the toxicity of these agents is greatly attenuated (A. Gray, J.
Morgan, "Liposomes in Haematology" in Blood Reviews, 1991, 5, 258-271).
Liposomes have been used in biological systems such as plasma extravascular
space like reticuloendothelial system to more access celluar uptake of
liposomes. Liposomes were loaded with amphotericin which is an effective
but toxic antifungal. Antitumor agents like adriamycine have also be
incorporated into liposomes. Vaccines and adjuvants as well as biological
response modifiers like lymphocines and so on were studyed in encapsulated
form. Liposomes are discussed in field of a gene transfert as vehicles.
N. Sakuragawa et al. report in Thrombosis Research 38, 681-685, 1985, 1988
Clinical Hematology 29 (5) 655-661, that liposomes containing factor VIII
have been prepared for oral administration to patients which are suffering
from von Willebrand's disease. The encapsulation was carried out by
dissolving the protein factor VIII concentrates in an aprotinin containing
solution and transferred into lecithin coated flasks. After drying the
flasks by rotation for 30 min under negative pressure liposomes were
formed which entrapped factor VIII concentrates. The liposome solution was
centrifuged yielding 40% of factor VIII entrapped in liposomes.
Another method for entrapment of drugs in liposomes is based on dehydration
- rehydration. This is described by C. Kirby and G. Gregoriadis in
Bio/Technology, November 1984, pages 979-984. In this preparation the
entrapments can be increased by using additional lipid. Disclosed is the
use of cholesterol as being of positive influence of the drug entrapment.
Since cholesterol is involed in the pathobio-chemistry of some disorders,
administration of cholesterol containing vesicles is not harmless at all.
Object of the present invention is to provide a method for encapsulating
biological structures, biopolymers and/or oligomers particularly those
being pharmaceutically active into lipid membrane vesicles giving higher
encapsulation of the respective substance. A further object is the
preparation of a formulation particularly a medicament having a higher
efficiency.
Surprisingly, one object of the invention is solved by a method for
preparation of vesicles loaded with biological structures, biopolymers
and/or -oligomers comprising the step of co-drying a fraction of
amphiphatic material and a fraction of biological structures, biopolymers
and/or -oligomers wherein said fraction of amphiphatic material is present
in an organic solvent which is miscible with water and said fraction of
biological structures, biopolymers and/or -oligomers is present in an
aqueous medium.
Liposomes can be classified according to various parameters.
For example, when size and number of lamellae (structural parameters) are
used than three major types of liposomes have been described:
Multilamellar vesicles (MLV), small unilamellar vesciles (SUV) and large
unilamellar vesicles (LUV). MLV are the species which form spontaneously
on hydration of dried phospholipids above their gel to liquid crystalline
phase transition temperature (Tm). Their size is heterogenous and their
structure resembles an onion skin of alternating, concentric aqueous and
lipid layers.
SUV are formed from MLV by sonication and are single layered. They are the
smallest species with a high surface-to-volume ratio and hence have the
lowest capture volume of aqueous space to weight of lipid.
A third type of liposome LUV has a large aqueous compartment and a single
(unilamellar) or only a few (oligolamellar) lipid layers.
Further details are disclosed in D. Lichtenberg and Y. Barenholz,
Liposomes: Preparation, Characterization, and Preservation, in Methods of
Biochemical Analysis, Vol. 33, pp. 337-462, as exemplified in FIG. 3.
As used herein the term "loading" means any kind of interaction of the
biopolymeric substances to be loaded, for example, an interaction such as
encapsulation, adhesion (to the inner or outer wall of the vesicle) or
embedding in the wall with or without extrusion of the biopolymeric
substances.
As used herein, the term "liposome" is intended to include all spheres or
vesicles of any amphiphatic compounds which may spontaneously or
non-spontaneously vesiculate, for example phospholipids where at least one
acyl group replaced by a complex phosphoric acid ester. The most of
triacylglycerol is suitable and most common phospholipids for the present
invention are the lecithines (also referred to as phosphatidylcholines
(PC)), which are mixtures of the diglycerides of stearic, palmitic, and
oleic acids linked to the choline ester of phosphoric acid. The lecithines
are found in all animals and plants such as eggs, soybeans, and animal
tissues (brain, heart, and the like) and can also be produced
synthetically. The source of the phospholipid or its method of synthesis
are not critical, any naturally occurring or synthetic phosphatide can be
used.
Examples of specific phosphatides are L-.alpha.-(distearoyl) lecithin,
L-.alpha.-(diapalmitoyl) lecithin, L-.alpha.-phosphatide acid,
L-.alpha.-(dilauroyl)-phosphatidic acid, L-.alpha.(dimyristoyl)
phosphatidic acid, L-.alpha.(dioleoyl)phosphatidic acid, DL-a(dipalmitoyl)
phosphatidic acid, L-.alpha.(distearoyl) phosphatidic acid, and the
various types of L-.alpha.-phosphatidylcholines prepared from brain,
liver, egg yolk, heart, soybean and the like, or synthetically, and salts
thereof. Other suitable modifications include the controlled peroxidation
of the fatty acyl residue cross-linkers in the phosphatidylcholines (PC)
and the zwitterionic amphiphates which form micelles by themselves or when
mixed with the PCs such as alkyl analogues of PC.
The phospholipids can vary in purity and can also be hydrogenated either
fully or partially. Hydrogenation reduces the level of unwanted
peroxidation, and modifies and controls the gel to liquid/crystalline
phase transition temperature (T.sub.m) which effects packing and leakage.
The liposomes can be "tailored" to the requirements of any specific
reservoir including various biological fluids, maintains their stability
without aggregation or chromatographic separation, and remains well
dispersed and suspended in the injected fluid. The fluidity in situ
changes due to the composition, temperature, salinity, bivalent ions and
presence of proteins. The liposome can be used with or without any other
solvent or surfactant.
Another important consideration in the selection of phospholipid is the
acyl chain composition thereof. Currently, it is preferred that it has an
acyl chain composition which is characteristic, at least with respect to
transition temperature (T.sub.m) or the acyl chain components in egg or
soybean PC, i. e., one chain saturated and one unsaturated or both being
unsaturated. However, the possibility of using two saturated chains is not
excluded.
The liposomes may contain other lipid components, as long as these do not
induce instability and/or aggregation and/or chromatographic separation.
This can be determined by routine experimentation.
A variety of methods for producing the modified liposomes which are
unilamellar or multilamellar are known and available:
1. A thin film of the phospholipid is hydrated with an aqueous medium
followed by mechanical shaking and/or ultrasonic irradition and/or
extrusion through a suitable filter;
2. dissolution of the phospholipid in a suitable organic solvent, mixing
with an aqueous medium followed by removal of the solvent;
3. use of gas-above its critical point (i. e., freons and other gases such
as CO.sub.2 or mixtures of CO.sub.2 and other gaseous hydrocarbons) or
4. Preparing lipid detergent mixed micelles then lowering the concentration
of the detergents to a level below its critical concentration at which
liposomes are formed (Lichtenberg, Barenholz, 1988).
In general, they produce liposomes with heterogeneous sizes from about 0.02
to 10 .mu.m or greater. Since liposomes which are relatively small and
well defined in size are preferred for use in the present invention, a
second processing step defined as "liposome down sizing" is for reducing
the size and size heterogeneity of lioosome suspensions.
The liposome suspension may be sized to achieve a selective size
distribution of vesicles in a size range less than about 5 .mu.m and
preferably to be .ltoreq.0.4 .mu.m. Liposomes in this range can readily be
sterilized by filtration through a suitable filter. Smaller vesicles also
show less a tendency to aggregate on storage, thus reducing potentially
serious blockage or plugging problems when the liposome is injected
intravenously. Finally, liposomes which have been sized down to the
submicron range show more uniform distribution.
Several techniques are available for reducing the sizes and size
heterogeneity of liposomes, in a manner suitable for the present
invention. Ultrasonic irradiation of a liposome suspension either by
standard bath or probe sonication produces a progressive size reduction
down to small unilamellar vesicles (SUVs) between 0.02 and 0.08 .mu.m in
size. Homogenization is another method which relies on shearing energy to
fragment large liposomes into smaller ones. In a typical homogenization
procedure the liposome suspension is recirculated through a standard
emulsion homogenizer until selected liposome sizes, typically between
about 0.1 and 0.5 .mu.m are observed. In both methods, the particle size
distribution can be monitored by conventional laser-beam particle size
determination.
Extrusion of liposomes through a small-pore polycarbonate filter or
equivalent membrane is also an effective method for reducing liposome
sizes down to a relatively well-defined size distribution whose average is
in the range between about 0.02 and 5 .mu.m, depending on the pore size of
the membrane. Typically, the suspension is cycled through one or two
stacked membranes several times until the desired liposome size
distribution is achieved. The liposome may be extruded through
successively smaller pore membranes, to achieve a gradual reduction in
lipsome size.
Centrifugation and molecular sieve chromatography are other methods which
are available for producing a liposome suspension with particle sizes
below a selected threshold less than 1 .mu.m. These two respective methods
involve preferential removal of large liposomes, rather than conversion of
large particles to smaller ones. Liposome yields are correspondingly
reduced.
The size-processed liposome suspension may be readily sterilized by passage
through a sterilizing membrane having a particle discrimination size of
about 0.4 .mu.m, such as a conventional 0.45 .mu.m depth membrane filter.
The liposomes are stable in lyophilized form and can be reconstituted
shortly before use by taking up in water.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 is a graph showing the in vivo induction of positive delayed type
hypersensitivity (DTH) by skin test membrane vaccine (STM), compared with
in vitro stimulation (stimulation index--SI) by STM (mixed lymphocyte
tumor culture--MLTC).
FIG. 2 shows a calibration curve constructed for the clotting assay of
factor IX.
FIG. 3 shows a scheme for carrying out a method according to the invention.
In a preferred embodiment the method of the invention comprises the steps:
a) solubilizing amphiphatic material in a polar-protic solvent being
miscible with water (fraction A), alternatively, dried lipids or lipid
mixture can be used in any form (powder, granular, etc.) directly,
b) solubilizing biopolymers and/or oligomers in an aqueous medium being
physiologically compatible, optionally having a salt content equivalent to
up to 5% by weight, preferably 15% by weight of sodium chloride solution
(fraction B)
c) mixing together the fractions A and B
d) drying the fraction obtained in step c) with a method retaining the
functional properties of said biological structures, biopolymers and/or
oligomers.
In general, the lipids mentioned above are suitable to be used for forming
lipid membrane vesicles. In particular saturated, unsaturated
phospholipids and combinations thereof are advantageously used according
to the method of the invention. Dimyristoyl phosphatidyl choline (DMPC)
and/or dimyristoyl phosphatidyl glycerol (DMPG) are used more preferably
for forming of the lipid vesicles. Preferably, the molar ratio of the
DMPC: DMPG is between 1:20 and 20:1.
According to the method of the invention the organic solvent being miscible
with water is a polar-protic solvent having solubilizing properties such
as for example aliphatic alcohols with lower number carbon atoms so long
as they mix with aqueous systems and do not affect adversely the
effectivity of the biological structures, biopolymers and/or -oligomers to
be encapsulated. Suitable alcohols are e. g. methanol, ethanol, propanol
and/or preferably t-butanol.
Biological structures to be encapsulated according to the invention are any
structures of higher order built up by various components and/or
substructures. Examples for these structures are whole cells, such as
natural or transformed B-cells, cell organelles, such as ribosoms or
mitochondriae. Virions or particles such as hepatitis B surface antigen
(HBsAg) particles. Biopolymers and/or -oligomers to be encapsulated
according to the invention are any substances having effects in human or
animal systems. Preferred are substances as proteins such as enzymes,
proenzymes, cofactors, such as those of the blood clotting system,
antigens, antibodies, factors the immune system such as complement
factors, peptides such as hormones, nucleotides and/or nucleic acids such
as genomic DNA for use in gene therapy, RNA such as MRNA, rRNA, tRNA,
antisense RNA and the like.
It is understood the skilled person that the amount of organic polar-protic
solvent miscible with water is strongly dependent on it interference with
the substance to be encapsulated to the liposomes. For example, for HBsAg
50% is tolerable while factor IX (which is a clotting-factor) is to be
encapsulated as an amount of approximately 30% of tert-butanol is
tolerable. This may strongly vary with the nature of the substance to be
encapsulated. For example, if factor IX which is a clotting factor is to
be encapsulated an amount of about 30% of tertiary butanol is tolerable,
whereas, factor VIII is much more sensitive to the impact of
tert.-butanol. In this case an amount of less than 10% of tert.-butanol is
preferred. The percentage of t-butanol in these examples is based on
percent by volume calculated for final concentration.
According to the method of the invention it is preferred to keep the
biopolymers and/or -oligomers in a medium having an ionic strength
corresponding up to about 5% sodium chloride concentration with or without
cryoprotectant which is a pharmaceutically acceptable agent such as
lactose, sucrose or trehalose, preferably the medium for solubilizing,
dissolving or dispersing the biological structures, biopolymers and/or
-oligomers is an aqueous solution of about 0.9% by weight sodium chloride
and/or an isoosmotic cryoprotectant.
According to the invention any method for drying is suitable so long as the
effectivity of the biological structures, biopolymers and /or -oligomers
are not affected adversely by the selected drying method. The function of
the biological structures, biopolymers and/or oligomers to be loaded are
mostly retained when mild drying conditions are selected. For example
removing of the solvents of the solution of the biological structures,
biopolymers and/or -oligomers and the lipids is favorably achieved by
drying under reduced pressure at slightly elevated temperatures at
maximum. The resistance of the active substances to be loaded depend
strongly on the stability the respective biopolymer and/or -oligomer. For
example, nucleic acids are more stable versus impact of heat on their
structure and function than proteins. The latter are more sensitive to
heat-denaturation. A very preferred method for co-drying of the fractions
according to the invention is the method of lyophilisation (freeze
drying). This method is a mild drying procedure for almost all of the
active biological structures, biopolymers and/or oligomers which become
liposomal loaded according to the invention.
According to the method of the invention the product obtained as described
above in dry form is taken up in an aqueous medium. Thereby, liposomes
formed become loaded with the respective biological structures,
biopolymers and/or oligomers. The system typically forms a dispersion.
According to the method of the invention a novel formulation is provided
comprising lipid membrane vesicles loaded with biological structures,
biopolymers and/or -oligomers. The formulation of the invention preferably
is in a solid state, which is available after the co-drying of fraction A
and B, eventually having other pharmaceutically acceptable vehicles and/or
adjuvants as well as other pharmaceutically active agents.
Another preferred embodiment of the formulation of the invention comprises
a solution of the fraction in an aqueous medium obtainable according to
the method of the invention. Preferably, the aqueous medium for taking up
the dry fraction of the formulation contains a balanced salt content in
order to adjust the conditions of the formulation in such a manner that
the aqueous solution thus obtained can readily be used as a medicament.
Typically, the formulation tends to form a dispersion after being taken up
into water.
Thus, the medicament of the invention is basically the formulation
obtainable by the method of the invention but being adapted to a way of
administration which is suitable for the treatment or prophylaxis of the
respective disease.
For example, the medicament of the invention can be administered by
topical, oral, intravenous, pulmonary, intraperitioneal, intranasal,
rectal, intraocular, buccal, subcutaneous and intramuscular ways of
application.
A method of treatment and/or prophylaxis of diseases by administering an
effective amount of the medicament according to the invention is provided.
It is understood by the skilled person that the dosage is depending on the
concentration of the effective substances as well as their efficiency.
According to the method of treatment and/or prophylaxis of the invention
preferably a dosage of up to 2,000 mg vesicles (e. g. phospholipid
liposomes)/kg body weight is administered to the patient. The accurate
dosage can vary dramatically. The variation, however, depends on e. g. the
type and efficacy of the substance encapsulated in the liposomes, the
efficiency of the encapsulation reaction itself (being high with the
method of the invention), the kind of administration and the like. The
respective parameters can be easily optimized by the person skilled in the
art and can be regarded as being routine experiments.
The invention is further explained by the following non-limiting examples.
EXAMPLE 1
Preparation of Samples of Anti-HBV Liposomal Vaccine
The following samples of vaccine, designated samples 1, 2, 3 and 4 were
prepared using the method of the invention.
Sample 1
A mixture of DMPC : DMPG in a molar ratio of 9:1 respectively was prepared
in tert.-butanol. An aqueous HBsAg solution such as 0.9% NaCl in 1:1 (v/v)
was added. The final HBsAg: phospholipids (w/w) ratio was 0.0015. The
solution was frozen and dried by lyophilisation. A dry powder was obtained
which was reconstituted before use with double distilled sterile
pyrogen-free water. Multilamellar liposomes were formed; loading
efficiency of HBsAg was 97%. "Empty liposomes" were prepared similarly by
mixing 1 vol of aqueous solution of 0.9%. NaCl with 1 vol of lipid
solution in tertiary butanol.
The extent of HBsAg exposure on the liposome surface of sample 1 and
liposome size was determined. It was found that the size of these lipsomes
was 4.5 .mu.m and the exposure of the antigen on the liposome surface was
tested. It was found that the titer of antibodies which was developed was
high and sufficient to protect against infection by HBV (see Table 1). The
titer was similar to that obtained in mice that were vaccinated with the
same antigen using aluminum hydroxide based vaccine except for the high
dose of injected antigen (2.5 .mu.g) in which the liposomal vaccine was
inferior: injection of this dose to mice in the control group stimulated
the highest titer of antibodies.
Sample 2
Liposomes loaded with HBsAg and "empty liposomes" were prepared as
described for sample 1. A group of seven Balb/c mice, six weeks old, were
vaccinated by 0.09 g HBsAg loaded in liposomes which were diluted with
"empty liposomes" and 0.9% NaCl. The final injection volume was 0.5
ml/mice, which included also 1 mg/kg mice of the immunomodulator MTP-PE in
POPC/DOPS (7:3 mole ratio) liposomes. After 35 days the level of anti-HBs
in the mice was measured. The titer of antibodies was twice the titer
which developed after injecting the same dose of antigen without MTP-PE
(sample 1).
Sample 3
Liposomes loaded with HBsAg and identical "empty liposomes" were prepared
as described for sample 1 with one difference in that the aqueous solution
used for lipid hydration also contained 5% lactose. The liposomes were
frozen and dried. A powder was obtained which was reconstituted before use
with sterile pyrogen-free bidistilled water. The liposomes were
characterized for their size, percentage of antigen loading and the extent
of antigen exposure on the liposome surface. The immunization efficacy of
the preparation was tested in Balb/c mice, six weeks old. The mice were
divided into three groups, five mice in each group, and the animals were
vaccinated using three doses of antigen: 0.09 .mu.g, 0.27 .mu.g, 0.81
.mu.g, respectively. Anti-HBs was measured after 35 days (see Table 1). A
high titer of antibodies was observed which should be sufficient to
protect against HBV infection.
Injecting this preparation in low doses of antigen (0.09 .mu.g) to mice
resulted in development of the highest titer of antibodies, compared with
the titer which was obtained with all other preparations including the
mice group which was vaccinated with the commonly used aluminum
hydroxide-based vaccine having identical HBsAg.
Sample 4
Liopsomes loaded with HBsAg were prepared as described for sample 3. Three
groups of five Balb/c mice, six weeks old, were vaccinated with four doses
of HBsAg at a level of 0.09 .mu.g, 0.27 .mu.g, 0.81 .mu.g, respectively.
The total injection volume was 0.5 ml/mice. The liposomes were diluted
with PBS only and not with "empty liposomes" and therefore the amount of
lipid varied and increased with increasing protein level. After 35 days
the mice were bled and their serum antibody titer was determined. The
results show a high titer of antibodies which should be sufficient to
protect against infection by HBV.
TABLE 1
__________________________________________________________________________
Summary of anti-HBs titer (mIU/ml) using the liposomal vaccine samples
described
in Example 1
.mu.m HBsAg injected
Sample No.
0.09 0.27 0.81 2.5
__________________________________________________________________________
1 52.4 .+-. 18.6
426.7 .+-. 206.3
4,953.2 .+-. 1,211.5
6,692.0 .+-. 854.5
2 106.1 .+-. 16.5
-- -- --
3 193.3 .+-. 69.1
1,664.6 .+-. 392.8
2,701.4 .+-. 203.6
--
4 55.0 .+-. 17.3
895.9 .+-. 384.6
1,527.7 .+-. 166.6
--
Control Alum-
40.0 .+-. 13.6
396.6 .+-. 73.1
6,749.3 .+-. 2,342.5
17,465.3 .+-. 2,967.0
based vaccine
__________________________________________________________________________
EXAMPLE 2
Stability of Liposomal HBsAc Vaccine After Storage at Various Temperatures
As described above hepatitis vaccines known in the art used aluminum
hydroxide as adjuvant and stabilizer. The disadvantage of the aluminum
hydroxide-based vaccines is that they cannot be frozen nor can they be
stored beyond 8.degree. C. These vaccines thus have to be stored between
2-8.degree. C. to maintain their efficacy.
There are three parameters to demonstrate stability of a vaccine under
different conditions:
1. Efficiency (measure immunogenicity).
2. Chemical stability (measssure hydrolysis of lipids; measure protein to
lipid ratio).
3. Physical stability (measure size of particle).
The stability of the vaccine was tested after storage at three temperature
(a) -20.degree. C., (b) 2-6.degree. C. and (c) room temperature.
The results obtained were as follows:
(a) The vaccine stored at -20.degree. C. was effective after 1 month or
more and was chemically and physically stable after 1.5 years and more.
(b) The vaccine stored at 2-6.degree. C. was effective after 1 month and
more and was chemically and physically stable after 1.5 years and more.
(c) The vaccine stored at room Temperature was chemically and physically
stable after 1.5 years or more.
These results demonstrate that the vaccine of the invention in form of
liposoms is stable over a wide temperature range.
Since the current hepatitis vaccines lose their immunogenicity during
freezing it is unexpected that the liposom-vaccine of the invention
retains its activity both during the freezing step of the freeze drying
process and also during storage of the vaccine below 0.degree. C.
Thus, the advantage of HBV vaccine of the invention is evident. It does not
need to be stored in a refrigerator and is not sensitive to freezing. The
distribution of such a vaccine is greatly simplified especially in third
world countries where the need for a vaccine against hepatitis B is
greatest; additionally a vaccine which may be frozen aids distribution in
countries such as Russia and China were the ambient temperature is often
below freezing.
Applicants have thus produced a novel liposomal based HBsAg vaccine which
is stable both below zero degrees and at room temperature, i. e. The
vaccine may be stored under suboptimal conditions.
EXAMPLE 3
Preparation and Characterization of Factor-IX-Loaded Liposomes
Two different methods of liposome preparation will be compared for
stability and Factor IX encapsulation.
(a) Dehydrated Reydrated Vesicles (DRV's)
(b) Lipid and drug co-solubilization in an organic solvent.
(a) Dehydrated Rehydrated Vesicles (DRV's)
Preparation of multilamellar vesicles loaded with Factor-IX by the DRV
method require the following steps: preparation of small unilamellar
vesicles (SUV's) in bidistilled water, mixing them with a solution of
factor IX previously dialyzed against amino acids and flash-frozen the
mixture. After lyophilization, multilamellar vesicles loaded with
Factor-IX were obtained by rehydrating the preparation with bidistilled
water, then stepwise saline is added, until the final liposomes
concentration was reached. At this point the multi-lamellar vesicles can
be sized by extrusion to obtain oligo-lamellar or small unilamellar
vesicles.
Rehydration of lyophilized material with minimal volume results in an
increase of the overall concentration of the factor. After liposomes are
formed the solution can be further diluted without affecting the loading
efficiency, and this is reflected in the concentration of the material
that is actually loaded. Since liposomes are osmotically active, losses of
material on exposure to hypotonic media during all manipulations
subsequent to hydrating were minimzed by dialyzing the Factor before
mixing with the SUV's to obtain a lower osmolarity in the liposome
interior during the rehydration step.
(b) Lipid and Drug Co-Solubilization in an Organic Solvent
In this preparation lipid solubilized in tert-butanol is mixed with an
aqueous solution of the factor to obtain an homogeneous solution. The
solution is frozen and the solvent removed by lyophilization.
Mulitlamellar vesicles loaded with Factor-IX are obtained by hydration of
the dry mixture, firstly in small volume of bidistilled water, then
stepwise with saline, until the final liposome concentration is reached.
At this point the multlamellar vesicles can be sized by extrusion to
obtain oligolamellar or small unilamellar vesicles.
Determination of Factor IX Activity
Factor IX activity was measured by a clotting assay. In this assay the
percent of factor IX activity can be determined by the degree of
correction obtained when a dilution of the tested sample is added to the
factor IX Deficient Plasma (purchased from Baxter Diagnostics Inc.). The
measuring instrument is called ACL-Automated Coagulation Laboratory from
Instrumentation Labortory (Italy).
A calibration curve was first constructed for the clotting assay of factor
IX, using appropriate dilutions of a stock solution of ca. 50 U/ml. FIG. 2
shows a good fit to a linear regression (R.sup.2 =0.989).
Liposomes containing factor IX were pelleted by centrifugation in an
Eppendorff centrifuge at 12,000 g for 10 min and the factor IX activitiy
was determined in the supernatants and pellet. The pellet was solubilized
prior analysis with Triton X-100. A concentration dependency on factor IX
activity with Triton X100 was found. 1% Triton X100 (final concentration)
caused a 50% loss of activity, while no loss was observed at 0.2%. In
general, the total activity of the factor was recuperated, namely, the
activity of the super-natants and pellet was always similar or even higher
than the inital activity of the preparation. The loading efficiency was
higher than 80%.
EXAMPLE 4
Melanoma Treatment of Human Patients by Liposomal Vaccine Containing
Allogenic Human Melanoma Vaccine Prepared Using Tertiary Butanol
Vaccine preparation
A mixture of DMPC:DMPG in a molar ratio of 9:1 respectively was dissolved
in tert-butanol in a 1:6.7 w/v ratio. The mixture was heated and stirred
until the lipids were dissolved. After sterile filtration, sterile water
was added to the organic mixture until a 1: 1 (v/v) ratio between the
tert-butanol and the water was reached. An aqueous solution of the
melanomic membrane mixture was added to a 1:750 protein : phospholipids
(w/w) ratio. This final mixture was divided in single doses of 1 g
phospholipids and each one was frozen and dried by lyophilization. A dry
powder was obtained and stored at -70.degree. C. Prior to application
liposomes were formed by rehydration in double distilled, sterile and
pyrogen-free aqueous solution containing 0.9% NaCl to obtain a liposome
dispersion of 10% phospholipid concentration. After reconstitution, this
liposomes had an average size of 1 .mu.m and an average phospholipid:
protein ratio of 765:1.
Treatment
A three arm randomized study for the treatment of melanoma by asi alone and
either systemic or regional interleukin-2.
Clinical and immunologial
Evaluation: eligible:
PTS with metastatic diseases;
ECOP PS 1-2: no previous
Immunother.; positive to 3/7
antigens (marieux).
Cimetidine
800 mg .times. 2/daily
4 weeks
Vaccine in liposomes
200 .mu.g protein/sile, (se),
at 2 sites, 10 weekly
immunizations
RANDOMIZATION
A Melanoma vaccine only-given on day 1. cimetidine 800 mg.times.2/daily.
B Melanoma vaccine only given on day 1, followed by IV IL-2, at one million
units/msq. on consecutive days 2, 3, 4. cimetidine 800 mg.times.2/daily.
C Melanoma vaccine given on day 1, followed by subcutaneous IL-2 at vaccine
site, concomitantly, IL-2 dose: 50.000 units/site at two sites, on days
1,2 & 3. Cimetidine 800 mg.times.2/daily.
__________________________________________________________________________
PAT
PROT
TREATMENT DISEASE
RESPONSE OUTCOME
__________________________________________________________________________
#1 A CIM + 4X V CC
Lung PD (2 m) Lung/Br/Liver
Dead (4 m)
#2 A CIM + 2X VACC
SC PD (2 m)Brain
Dead (6 m)
#3 A CIM + 5X VACC
LN/LIVER
PD (2 m) LN/Liver
Alive (6 m)
#4 A CIM + 5X VACC
LN/Liver
PD (2 m) LN/Liver
Alive (6 m)
#1 B CIM + 10X VACC + IL2 S
Liver PD (3.5 m)/Liver/Bone
Dead (8 m)
#2 B CIM + 10X VACC + IL2 S
LN/Bone
PD (4 m) LN/Bone
Alive (16 m)
#3 B CIM + 10X VACC - IL2S
LN PD (4 m) LN/Lung
Alive (13 m)
#4 B CIM + 6X VACC + IL2 S
LN PD? (sepsis) Dead (2.5 m)
#1 C CIM + 10X VACC + IL2 R
LN/Lung
CR (8 m) LN/Lung
Alive (13 m) NED
#2 C CIM + 10X VACC + IL2 R
LN CR (9 m) LN Alive (13 m) NED
#3 C CIM + 10X VACC + IL2 R
LN/Liver
PD (3 m) LN/Liver
Dead (6 m)
#4 C CIM + 10X VACC + IL2 R
LN/Lung
PR (4 m) LN(PR)/Lung (CR)
Alive (12 m) Surg
NED
#5 C CIM + 10X VACC + IL2 R
SC/Liver/Bone
PR (5 m) SC(PR)/Liver (CR)
Dead (9 m) PD Brain
#6 C CIM + 10X VACC + IL2 R
SC/Lung
MixR (5 m) Alive (11 m) IPL NED
SC (PD)/Lung (CR)
#7 C CIM + 7X VACC + IL2 R
SC/LN/Liver
PD (3 m) SC/Liver
Dead (5 m)
#8 C CIM + 5X VACC + IL2 R
SC/Lung/Liver
PD (1.5 m) SC/Liver/Lung
Alive (2 m)
__________________________________________________________________________
CR=complete response
PR=partial response
SC=stable condition
m=month
(1) Allogeneic human melanoma vaccine was prepared from membranes of six
melanoma cell lines which express both class I and II MHC antigens and
MAAs (by R24 and P97) MoAbs);
(2) Membranes were loaded in liposomes consisting of DMPC: DMPG in a 9:1
molar ratio, were tested for sterility, pyrogenicity and tumorigenicity in
nude mice;.
(3) 16 PTS, (patients) were treated by vaccine (FIG. 1): 4--vaccine only
(A); 4--vaccine+systemic IL2 (B); and 8--by vaccine+low-dose, regional (C)
IL2;
(4) Clinical responses (regression of metastases) were observed in 5 of 8
PTS in arm C of the protocol;
(5) The above clinical responses correlated with de novo induction-of
cutaneous DTHI to membrane vaccine preparation (STM) and in vitro MLTC
(proliferative) responses to STM;
(6) Augmented cytolytic responses against melanoma cell lines were observed
in the majority of vaccine-treated PTS, but these were not MHC-restricted,
nor did they show any correlation with clinical responses;
(7) Selective anti-melanoma cytolytic responses following IVS (in vitro
stimulation) were observed when 18 h--instead of 4 h assay was used,
suggesting CD4, T-cell response, also corroborated by surface markers
study;
(8) In parallel patients were vaccinated with the same antigens given as
alumm based vaccine without any response.
EXAMPLE 5
Candidemia Treatment in Mice by Liposomal Vaccine Containing Candida
Ribosomes
Vaccine Preparation
DMPC: DMPG at a 9:1 molar ratio were dissolved in tert-butanol in a 1:10
(w:v) ratio and the lipid mixture was pre-warmed to dissolve the lipids
completely. An aqueous ribosomal mixture containing 1.5 mg ribosomes/ml
(determined by Orcinol) was added to the lipids at a 1:100 w/w final
ratio. In some cases Lipid-A was added at this stage as an adjuvant in a
1:1,000 lipid-A to phospholipids molar ratio. This suspension was frozen
and lyophilized in aliquouts of 0.5 g phospholipids and the dry powder was
stored at -20.degree. C. Prior application liposomes were formed by adding
two aliquots of 0.5 ml volume of double distilled, sterile and pyrogen
free aqueous solution containing 0.9% NaCl.
Treatment
Four groups of five Balb/c mice, six weeks old, were vaccinated with a one
single dose of 100 .mu.g ribosomes. Two weeks later a booster injection
was given and twenty eight days after the first immunization the mice were
challenged by intravenous infection with 10.sup.4 Candida albicans cells.
Group a: buffer (TMB) and IFA (incomplete Freund adjuvant).
Group 2: ribosomal mixture and IFA
Group 3: liposomes containing ribosomes
Group 4: liposomes containing ribosomes and lipid-A.
This experiment was repeated twice and the results are summarized in the
following table.
TABLE 3
______________________________________
Group 1
Group 2 Group 3 Group 4
______________________________________
Mortality 6/9 2/10 0/10 0/10
Percentage 67% 20% 0% 0%
______________________________________
EXAMPLE 6
Preparation of Liposomes Containing Anti-haemophilic Factor IX
Liposomes Preparation
Purified egg yolk phosphatidylcholine was dissolved in tert-butanol at
various ratios and the mixture was slightly warmed until the phospholipid
was dissolved. Double distilled sterile, pyrogen free water was added
until the desired ratio between the organic solvent and the water was
reached. An aqueous solution of salt free Factor IX (OCTANYNE.RTM.
adjusted pH 7.4 was added to the suspension under continuous mixing and
subsequently lyophilized. The ratio of the total protein to phospholipid
was 1:400 (w/w). The dry mixture was stored at 40.degree. C. Liposomes of
1 .mu.m average size were prepared by hydrating the powder with aliquots
of sterile, pyrogen-free double distilled water and mixing well between
the additions. The last addition consisted of saline to raise the salt
concentration to isosmotic conditions.
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